ABSTRACT The kidney is the major source of erythropoietin (EPO) production in adults. Loss of EPO-producing cells in chronic kidney disease (CKD) patients causes depressed red blood cell production and is a significant contributor to morbidity. Although CKD patients can be treated by systemic administration of EPO, this leads to sporadic and excessive peaks in hormone availability, which may provide growth stimuli to certain tumors. In addition, administration of recombinant EPO or EPO stimulating agents (ESAs) to CKD patients is associated with increased risk of cardiovascular events including cardiac arrest and stroke. It is not clear whether these morbidities are caused by excessive red blood cell number (polycythemia) stimulated by EPO or off-target effects of EPO itself. Finally, the cost of administering recombinant EPO to CKD patients now represents a significant proportion of the Medicare budget. Therefore, there are strong clinical and economic incentives for deriving an induced pluripotent stem cell (iPSC)-derived EPO-producing cell that could be used to treat anemic CKD patients. We propose to define conditions to generate EPO-producing cells by directed differentiation through recapitulating the developmental process that specifies this cell type within the developing kidney. Genetic analyses have revealed that the renal EPO-producing cell (REPC) is a stromal fibroblast located around proximal tubules in the outer medulla of the kidney. REPCs derive from the Foxd1-expressing stromal precursor population that is present only during the period of active kidney development. The REPC population has been difficult to identify because EPO is only expressed upon hypoxia, and other markers to specifically identify these cells are lacking. A deeper understanding of the molecular identity of the REPC is essential to define a target state for directed differentiation of iPSCs. We therefore propose to: 1. Define the molecular profile of normoxic REPCs through genetic labelling and isolation, and 2. Use EPO-reporter iPSCs to test the capacity of organoid differentiation protocols to generate hypoxia responsive EPO-producing cells. Because the EPO-producing cell has only been spatially defined in the mouse, we see this tandem approach using both mouse genetic tools and organoids differentiated from genetically modified human iPSCs as essential. The experiments are technically feasible, and our group has expertise in stromal cell biology, which will be essential to developing differentiation conditions. In the short term we would aim to functionally test these cells by encapsulating them in microspheres and implanting them into the abdominal cavities of mice subjected to nephrectomy. In the long term, we would aim to incorporate hypoxia-responsive, EPO-producing cells into synthetic kidney tissue destined for clinical translation.